>: Promega's Taq doesn't like Cetus buffer, and the presence of detergent in
>: the Promega buffer may be the reason, altho I haven't tried to add
>: detergent to the Cetus stuff.
>>Jack Kramer wrote:
>:I had similar problems with the Promega Taq with <Promega's 10X
>:buffer. The Cetus Taq had similar problems in the same buffer.
>:Switching to the Cetus buffer cured the problem. With this buffer,
>:or modifications of it which <i make up myself now, I get identical
>:results with either Taq. Try Promega Taq in storage buffer B and make
>:up your own reaction buffers - saves bundles of money!
>
As a producer I have tried various buffers but cannot find one that satisfies
everyone. Some people swear by the Promega recipe, other prefer Cetus. I can
give the same enzyme to several labs they only get it to work in their hands
with a certain buffer. I'm convinced that this is due to the source of
dNTP's/water/DNA/primers etc they use. However one buffer that seems to have
universal acceptance is as follows:
10xBuffer for Taq pol from YT1, B and Tth strains.
750mM Tris-HCl pH 8.8 @25C, 200mM AmmSO4, 15mM MgCl2, 0.1%(w/v) Tween 20.
If you want Mg separate then take pH up to 9.0 or even make up a set of buffers
with different Mg and/or pH combinations. ie Mg in 0.25mM difference and pH in
0.25 difference. pH can have a marked effect. I have tried using Tricine which
buffers better than Tris but the Mg optimum is higher, although it isn't a
sharp peak.
Make up as much as you need then dispense into screwcap eppendorfs (1.25ml in
1.5ml vials) and autoclave standing up! Use AR reagents and best water
available. Store as you like.
Make sure that any dNTP's you use are pH'd otherwise they might take the
reaction pH down.
Duncan
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Duncan Clark | Internet: duncan at genesys@demon.co.uk
GeneSys Ltd. | Compuserve: 100015.1406.compuserve.com
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